Abstract

To delineate further the pathway of pepsin-catalysed reactions, three types of experiments were performed: (a) the enzyme-catalysed hydrolysis of a number of di- and tri-peptide substrates was studied with a view to observing the rate-determining breakdown of a common intermediate; (b) the interaction of pepsin with several possible substrates for which ‘burst’ kinetics might be expected was investigated; (c) attempts were made to trap a possible acyl-enzyme intermediate with [14C]methanol in both a hydrolytic reaction (with N-acetyl-l-phenylalanyl-l-phenylalanylglycine) and in a ‘virtual’ reaction (with N-acetyl-l-phenylalanine) under conditions where extensive hydrolysis or 18O exchange is known to occur. It is concluded that (i) intermediates in pepsin-catalysed reactions (aside from the Michaelis complex) occur subsequently to the rate-determining transition state, and (ii) an acyl-enzyme intermediate, if such is formed, cannot be trapped with [14C]methanol in these systems.